The alpha helix is the most common secondary structure in the native state of proteins, involving nearly one third of the amino acids present. This project seeks to identify the interactions responsible for helix stabilization, and to determine at what stage(s) in folding a helical structure is important. The free energies of individual interactions involved in stabilizing helical structure will be determined, using synthetic model peptides and measurement of hydrogen exchange rates by 1/H NMR. The interactions to be monitored include the helical propensity of a given side chain, in various contexts; side chain-side chain interactions, including charged and hydrophobic side chains, and helix capping interactions. The role of helix stabilizing interactions in the native state and in folding intermediates of a helical protein will be investigated, using a series of mutant myoglobins and apomyoglobins. Methods include spectroscopic definition of mutant proteins in the presence of heme, using absorbance and CD. The stability of the mutant myoglobins will be determined by thermal and solvent induced unfolding. Structures of interesting proteins will be further investigated using a new surface fingerprinting technique as well as standard techniques. The structure and stability of compact, molten globular intermediate states in apomyoglobin and the protein with heme and dyes such as ANS which bind at the heme pocket will be investigated. Specific questions to be answered include: (i) What interactions control the stability of isolated alpha helical structure? (ii) Do these play a role in early intermediates in folding a protein? (iii) How is the helix content of intermediates in myoglobin folding established? (iv) Can the globin fold be redesigned using mutants at helix-helix cross-overs and interfaces? The results should provide a detailed picture of alpical structure in models and in folding of helical proteins.